PROJECT SUMMARY Super-resolution microscopy (nanoscopy) enables the visualization of live cells at resolutions that exceed the diffraction limit of light, far beyond the capability of ordinary light microscopes. Observing living cells at resolutions <20 nm reveals details of organelle structure, function, and dynamics that deepen our fundamental understanding of cell biology. While there has been significant progress in the engineering, design, and development of improved nanoscopy hardware, the field is limited by the fuel?the dyes that support the ever more sophisticated requirements of both Stimulated Emission Depletion (STED) and Single Molecule Switching (SMS). These requirements, which include brightness, photostability, cell-permeability, and for SMS: the ability to ?blink,? represent real chemical challenges that currently limit the full potential of these methodologies. Recently, the Silicon Rhodamine (SiR) scaffold has been shown to exhibit many promising characteristics for live-cell super-resolution imaging (SiR-CO2H) such as cell-permeability, photostability, and the ability to blink spontaneously (HMSiR) but has in no way been fully optimized. Work in the Schepartz Lab addresses the aforementioned challenges from a chemical perspective through the design of probes and fluorophores that are tailored to overcome these challenges while meeting the needs of nanoscopy techniques. These efforts include the development of a lipid probe system for super-resolution imaging of the Golgi (published work) and Endoplasmic Reticulum (ER) (unpublished work). This work was performed in close collaboration with the Rothman, Toomre, and Bewersdorf labs in Yale Cell Biology. The proposed research seeks to develop new and improved fluorophores that will take full advantage of cutting- edge nanoscopy techniques available at Yale and elsewhere, providing new tools to advance our understanding of biology and medicine.